The proposed experiments are directed towards understanding in molecular terms the mechanism whereby a transposable element, the kanamycin resistance determinant Tn5 is excised from its sites of insertion and transposed to new sites in the genome of Escherichia coli. (1) The specificity of insertion site selection in the bacterial genome as a whole will be studied by DNA-DNA hybridization to separated fragments of the E. coli chromosome. The randomness of insertion will be estimated by fine structure genetic mapping of sites of insertion of Tn5 in the lac and trp operons. (2) The deletion endpoints resulting from imprecise excision of the Tn5 element from sites in the lacZ gene will be mapped genetically. (3) To determine if genes present in Tn5 are necessary for transposition, deletions within the Tn5 element will be generated in vivo, and also by use of a colE1 plasmid vector to clone specific DNA fragments generated by digestion with restriction endonucleases. If these deletions are defective in transposition, amber and temperature sensitive mutations in tranposition genes will be selected. (4) The kinetics of transposition of Tn5 from an infecting lambda Tn5 phage to an F' plasmid will be measured using F' x F-interrupted matings. Attempts to increase the frequency of transposition by stimulating N lambda promoted readthrough transcription of the Tn5 element after lambda Tn5 phage infection will be undertaken. (5) Finally, proteins specified by Tn5 DNA templates in vitro and in vivo will be characterized, and the activity of proteins which might function in transposition by binding Tn5 DNA sequences specifically will be sought.